Menopause is a biological process in which a woman's ovaries reduce but do not completely stop their production of female sex hormones. Menopause is diagnosed when menstruation ceases permanently. Changing levels of female sex hormones that precede and postdate menopause often cause a variety of symptoms. Common health issues related to the menopausal transition and menopause include: irregular periods, hot flashes, increased risk of vaginal and/or bladder infection, urge incontinence, stress incontinence, fatigue, depression, loss of muscle mass, increased fat tissue, thinning and loss of skin elasticity, loss of bone tissue, impaired cognition, and an increased prevalence of cardiovascular disease.
One therapy used to combat symptoms associated with changing levels of female sex hormones is hormone replacement therapy (HRT). HRT is the administration of the female hormones including estrogen, progesterone, and androgens. One form of HRT is estrogen replacement therapy (ERT), which is the administration of estrogen alone. It is believed that HRT and ERT help in relieving symptoms of menopause and can be used to combat osteoporosis and to prevent the early onset of heart disease, two conditions often associated with postmenopause.
The long term benefits of HRT and ERT, however, remain questionable due to concerns regarding safety and efficacy. For example, HRT and/or ERT may increase an individual's risk of developing cancer. Specifically, it has been reported that prolonged exposure of the breast tissue to estrogen for five or more years of exogenous ERT is linked to an increased risk of breast cancer (Collaborative Group on Hormonal Factors in Breast Cancer, Lancet, 350:1047-1059).
It is believed that estrogen biosynthesis and function play an important role in the pathogenesis of certain cancers. This is true for breast cancer and is clinically correlated with the prevalence of breast cancer in women with evidence of increased endogenous estradiol synthesis. Thus, women with increased mammographic breast density, a clinical manifestation of excessive breast tissue estrogen synthesis, are at a higher risk of breast cancer (Boyd, N. F. et al., “Heritability of mammographic density, a risk factor for breast cancer,” N. Engl. J. Med., 347(12):886-94 (2002)) as are women with levels of serum estradiol levels in the upper quartile, than postmenopausal women with normal serum estradiol levels in the lower quartile (Cauley, J. A. et al., “Elevated serum estradiol and testosterone concentrations are associated with a high risk for breast cancer. Study of Osteoporotic Fractures Research Group,” Ann. Intern. Med., 130(4 Pt 1):270-7 (1999)).
The extent to which exogenous estrogen contributes to this pathology is not precisely established, but may be influenced by an individual's predisposition to cancer, by pathways of estrogen metabolism and catabolism, or by the route and dose of ERT or HRT. Breast cancer has well-defined histological criteria, but the etiology and pathogenesis of the cancer is varied for each histological type. In this context, the diagnosis of ‘breast cancer’ can be thought of as a syndrome, with cancers of varying degrees of differentiation, in which estrogen biosynthesis and function play a role. This is true for both endogenous estrogen production and estrogen replacement therapy (ERT) (Clemons, M. and P. Gross, “Estrogen and the risk of breast cancer,” N. Engl. J. Med. 344:276-285 (2001)). Significantly, the HRT-related increase in breast cancer is most frequently associated with invasive breast cancer having a favorable histology (Gapstur, S. M. et al., “Hormone replacement therapy and risk of breast cancer with a favorable histology: results of the Iowa Women's Health Study,” JAMA, 281(22):2091-7 (1999)) and prognosis (Gajdos, C. et al., “Breast cancer diagnosed during hormone replacement therapy,” Obstet. Gynecol., 95(4):513-8 (2000)).
In postmenopausal women, estrogen synthesis and metabolism takes place primarily in non-ovarian sites: adipose tissue, muscle, and liver. As illustrated in FIG. 1, the amount and type of estrogen synthesized is governed by three enzyme systems: (1) cytochrome p450 17α-hydroxylase/C17-20 lyase activity is responsible for the production of dehydroepiandrosterone (DHEA) (from pregnenone); (2) 3β-hydroxysteroid dehydrogenase (3β-OHSD)-Δ5,4-isomerase catalyzes the conversion of DHEA to androstenedione; (3) p450 aromatase regulates the conversion of androstenedione to estrone, and testosterone to estradiol; while (4) 17β-hydroxysteroid dehydrogenase (17β-OHSD) modulates the bi-directional formation of androstenedione to testosterone, and estrone to the more potent estradiol.
The 17α-hydroxylase enzyme and the aromatase enzyme are both genetically controlled by cytochrome CYP17 and cytochrome CYP19, respectively. It has been found that women with CYP17 polymorphism have high serum estradiol values (Hairman, C. A. et al., “The relationship between a polymorphism in CYP17 with plasma hormone levels and breast cancer,” Cancer Res., 59:1015-1020 (1999)). The aromatase gene may, under certain biological conditions, act as an oncogene within breast tissue (Siegelmann-Danieli, N. and K. H. Butetow, “Constitutional genetic variation at the human aromatase gene (Cyp19) and breast cancer risk,” Br. J. Cancer, 79:456-463 (1999)). Also, 17β-OHSD levels are higher in breast cancer compared with normal breast tissue, and may influence the course and progression of the disease by elevating local estrogen production (Vermeulen, A. et al., “Steroid dynamics in the normal and carcinomatous mammary gland,” J. Steroid Biochem., 25:799-802 (1986)).
The metabolism of estrogen follows pathways that result in either estrogenic or non-estrogenic metabolites. Estrogen catabolism takes place in two stages: hydroxylation and methylation. Estrone and estradiol are hydroxylated to the following: 2-hydroxyestrone (2-OHE1), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestrone (4-OHE1), 4-hydroxyestradiol (4-OHE2), 16-hydroxyestrone (16-OHE1), and 16-hydroxyestradiol (16-OHE2). Women who preferentially metabolize their endogenous estrogens via the 16α-hydroxylation pathway (versus 2α-hydroxylation) are at higher risk of breast cancer (Ursin et al., “Urinary 2-hydroxyestrone/16alpha-hydroxyestrone ratio and risk of breast cancer in postmenopausal women,” J. Natl. Cancer Inst., 91:1067-1072 (1999)) as are women with elevated 4-hydroxylase activity in their breast tissue (Lieber, J. G. et al., “Adenovirus-mediated urokinase gene transfer induces liver regeneration and allows for efficient retrovirus transduction of hepatocytes in vivo,” Proc. Natl. Acad. Sci. U.S.A., 92(13):6210-4 (1995)). The hydroxylation of estrogen is genetically controlled: for example, the CYP1A1 gene, which encodes cytochrome p450 CYP1A1, inhibits or downgrades the activity of the 2α-hydroxy pathway, thereby diminishing the concentration of the benign protective 2-hydroxy estrogens in favor of the biologically potent 16-hydroxyestradiol metabolite (Huang et al., “Breast cancer risk associated with genotype polymorphism of the estrogen-metabolizing genes CYP17, CYP1A1, and COMT: a multigenic study on cancer susceptibility,” Cancer Res., 59:4870-4875 (1999)), while 4-hydroxylation is catalyzed via CYP1B1 (Hayes et al., “17 beta-estradiol hydroxylation catalyzed by human cytochrome p450 1B1,” Proc. Natl. Acad. Sci. U.S.A., 93:9776-9781 (1996)).
The 2- and 4-hydroxy metabolites are methoxylated via catechol-O-methyltransferase (COMT) activity into anticarcinogenic metabolites with little or no estrogen receptor binding affinity. There are significant variations among women in COMT activity. It has been postulated that women with lower COMT activity may be at higher risk of estrogen-associated breast cancer due to decreased formation of the anti-tumorugenic 2-methoxyestradiol and the retarded inactivation of the pro-angiogenic 4-OHE2 metabolite (Zhu, B. J. and A. H. Conney, “Functional role of estrogen metabolism in target cells: review and perspectives,” Carcinogenisis, 19:1-27 (1998)). Conversely, upregulation of 2-methoxyestradiol synthesis inhibits the proliferation of human breast cancer cell lines (in both estrogen receptor positive or negative breast cancer cells) and is a potent inhibitor of angiogenesis. Thus, factors, including hormone therapy, that either increase or decrease this estrogen metabolite, could have a profound influence on the pathogenesis of breast cancer (Chushman, M. et al., “Synthesis, antitubulin and antimitotic activity, and cytotoxicity of analogs of 2-methoxyestradiol, an endogenous mammalian metabolite of estradiol that inhibits tubulin polymerization by binding to the colchicine binding site,” J. Med. Chem., 38:2041-2049 (1995); Fotsis, J. et al., “2-methoxyestradiol, an endogenous estrogen metabolite, inhibits angiogenesis and suppresses tumor growth,” Nature, 368:237-239 (1994)). Postmenopausal women with a variant COMT allele are at increased risk of breast cancer (Lavigne et al., “An association between the allele coding for a low activity variant of catechol-O-methyltransferase and the risk for breast cancer,” Cancer Res., 57:5493-5497 (1997)). In short, metaboliles such as 16α-OHE1 are powerful estrogen agonists whereas metabolites such as 2-OHE1 act as estrogen antagonists. It is unclear whether the absolute concentration of these metabolites determines the eventual biological effect on breast tissue, or whether it is the ratio between the two metabolites (2-OHE1/16α-OHE1) that is of greater importance (Lippert, T. H. et al., “Estradiol metabolism during oral and transdermal estradiol replacement therapy in postmenopausal women,” Horm. Metab. Res., 30(9):598-600 (1998)). The same may be true for the absolute amounts or ratios of other estrogen metabolites that may have competing oncogenic or anti-carcinogenic activity i.e., activity due to polymorphism in the COMT gene.
Estrone sulfate is formed by peripheral conversion from estradiol and estrone. Studies have documented that 65% of the estradiol and 54% of the estrone produced are converted to estrone sulfate (Ruder et al., “Estrone sulfate: production rate and metabolism in man,” J. Clin. Invest., 51:1020-1023 (1972)). In absolute terms, the circulating level of estrone sulfate is 10-25 times that of estrone and estradiol. Estrone sulfate thus functions as a major reservoir for estrone and estradiol (Lobo, R. A., “Androgen excess and the infertile woman,” Obstet. Gynecol. Clin. North Am., 14:143-167 (1987)). Sulfatases in various tissues (and especially the breast) reduce the inactive estrone sulfate to estrone. Estrone is subsequently reduced to estradiol by 17β-OHSD.
Sulfatase (and 17β-OHSD) activity in breast tissue is quantitatively greater than that of aromatase, and is especially high in women with breast cancer (Pasqualini, Jr. et al., “Concentrations of estrone, estradiol, and estrone sulfate and evaluation of sulfatase and aromatase activities in pre- and postmenopausal breast cancer patients,” J. Clin. Endocrinol. Metab., 81:1460-1464 (1996)). The persistence of significant levels of testosterone in postmenopausal women is due to the peripheral conversion of the adrenal androgen precursor DHEA-S (dehydroepiandrosterone sulfate) to DHEA (dehydroandrosterone) via sulfatase activity. Testosterone is bio-converted to estradiol (Zumoff B. et al., “Twenty-four-hour mean plasma testosterone concentration declines with age in normal premenopausal women,” J. Clin. Endocrinol. Metab., 80:1429-1430 (1995)). The contribution of the DHEA-S/DHEA prohormones to breast cancer is unknown, but both are significant substrates for breast tissue synthesis of estradiol. Excess aromatization of testosterone to estradiol is thought by some researchers to correlate significantly with the risk of hormone-induced breast cancer. Others believe that androgens normally inhibit mammary epithelial growth (Dimitrakakis, C. et al., “Androgens and mammary growth and neoplasia,” Fertil. Steril., 77(Suppl. 4):26-33 (2002)).
Although prolonged exposure of the breast tissue to estrogen is linked to an increased risk of breast cancer, identification of the individual at risk is problematic. Measuring serum estradiol levels provides some guidance, even though peripheral estradiol blood levels are substantially lower than breast tissue—and especially breast cancer tissue—levels (Pasqualini, Jr., et al., J. Clin Endocrinol Metab (1996)). The estradiol serum values associated with breast cancer risk are also much lower than previously accepted ‘normal’ postmenopausal estrogen blood level (Cauley, J. A. et al., Ann. Intern. Med., (1999)). It is now possible to predict the estrogen receptor status of breast tumors using gene expression data with 90% accuracy (West, M. et al., “Predicting the clinical status of human breast cancer by using gene expression profiles,” Proc. Natl. Acad. Sci., 98:11-62-67 (2001)). In addition, for those who are found to be estrogen receptor positive, the individual breast tissue estrogen metabolite pathway, and the potential for breast cancer, could be identified by establishing the women's cytochrome p450 characteristics (Nebert, D. W. and D. W. Russell, “Clinical importance of the cytochromes P450,” Lancet, 360:1155-1162 (2002)).
Genotyping of women at risk for breast cancer is a promising new approach. Polymorphism of cytochrome CYP17 (resulting in raised estrogen concentrations) and cytochrome CYP1A1 activity (inhibition of 2α-OHE1 and 2α-OHE2) and decreased COMT activity (decreased methoxylation of the potent OHE1 metabolite) were, in one study, associated with an increased risk of breast cancer (Huang et al., Cancer Res. (1999)). Advances in gene technology now permit the measurement of candidate gene polymorphisms that alter in-situ estrogen synthesis in liver (COMT, CY1B1, CY1A1), in breast tissue (CYP1a, CYP1B1, CYP1A1), and the adrenal glands/ovaries (CYP17), and which will then allow for the selective use of microassay analysis to assess breast tissue expression of genes that metabolize hormones and which are clear indicators of the breast tissue's response to estrogen. This approach adopts the documented value of microarray analysis that has been developed as a predictor of the outcome of disease in young women with breast cancer (van de Vijver et al., “A gene-expression signature as a predictor of survival in breast cancer,” N. Engl. J. Med., 347:1999-2009 (2002)), but allows for the early identification of women with high pro-carcinogenic estrogen metabolic profiles, and hence, the ability to selectively prescribe chemopreventive therapies such as sulfatase or aromatase inhibitors, and/or selective estrogen receptor modulators.
The impact of prescribed ERT, and its effect on the breast, is additive to the breast tissue pretreatment hormonal milieu. The ‘dose’ of estrogen that might initiate or promote the transformation of normal breast tissue to cancer is not known. However, there are women whose estrogen-sensitive target organs display an exaggerated response to physiological levels of estradiol.
The route of ERT may be important in women with certain estrogen metabolic polymorphism. An oral daily dose of 1 mg 17β-estradiol is considered to be equivalent, in terms of clinical efficacy, to biweekly 50 μg transdermal estradiol. This dose comparison may be misleading, however, because it does not account for large diffusion in estradiol metabolite concentrations nor does it account for the increase in sex hormone-binding globulin (SHBG) after oral, but not transdermal, ERT (Vehkavaara, S. et al., “Differential effects of oral and transdermal estrogen replacement therapy on endothelial function in postmenopausal women,” Circulation, 102:2687-2693 (2000)). The long term consequences of this SHBG-binding variance has not been documented in clinical trials, but does account for many instances of ‘non-response to adequate ERT’ experienced by women in clinical practice.
Because of increased SHBG binding (and possibly other factors), more estrogen is needed after oral ERT to achieve a clinical response equivalent to that obtained by non-oral ERT. Only 5% of oral estradiol is bioavailable following the first-pass hepatic metabolism of oral estradiol. There is only minimal first-pass metabolism of non-oral estradiol (Kuhnz et al., “Pharmacokinetics of estradiol, free and total estrone, in young women following single intravenous and oral administration of 17 beta-estradiol,” Arzneimittelforschung, 43:966-973 (1993)). Over time, the difference in hepatic modification of estradiol between the oral and transdermal routes is lessened.
When the gastric transit time and the enterohepatic recirculation of estrogen are factored in, oral estradiol therapy results in supraphysiological levels of estrone sulfate. This is reflected in a recent study summarized in the following Table 1 which compares the blood levels of the pro-hormone estrone sulfate, following equivalent doses of either oral or transdermal estradiol (Slater, C. C. et al., “Markedly elevated levels of estrone sulfate after long-term oral, but not transdermal, administration of estradiol in postmenopausal women,” Menopause, 8:200-203 (2001)). Normal menstrual cycle levels of E1S were at 2-3 ng/mL. The clinical significance of prolonged supra-physiological levels of estrone sulfate is not known. Depending on the breast tissue sulfatase and 17β-OHSD activity, high concentrations of local breast tissue estradiol can be anticipated. The amount of estrone sulfate absorbed and metabolized after oral ERT may vary with the composition of the oral estrogen prescribed. Approximately 45% of the total dose of conjugated equine estrogens (CEE), 75-85% of esterified estrogen and 75% of piperazine sulfate is composed of estrone sulfate ((Slater et al, 2001, Menopause 8:200). Although a portion of estrone sulfate is hydrolyzed to estrone in the intestine, the unconjugated estrogen is reconjugated in the liver to the sulfate. Estrone sulfate is also subject to hepatic recirculation.
TABLE 1Estrone Sulfate (E1S) ng/mL Following Oral v. Transdermal EstradiolEndogenous E1S 10-25 > E1 and E2EstradiolEstradiolEstradiolPlaceboTest Interval1 mg (oral)50 mcg (TD)100 mcg (TD)patchB0.800.700.800.80125.001.602.301.00239.001.803.201.20P<0.01<0.05<0.05N/S
Supraphysiologic breast tissue levels of estradiol, apart from directly inducing proliferation of ductal and epithelial elements of the breast, could via regulatory mutation alter the expression of a gene that leads to either a loss of function or to a positive expression in a tissue, in which it was previously silent (Guttmacher, A. E. and F. S. Collins, “Genomic medicine—a primer,” N. Eng. J. Med., 347:1512-1520 (2002)).